Dengue virus (DENV) is a single-stranded RNA flavivirus that causes dengue fever, the most prevalent arthropod-borne viral illness in humans (NIAID Category A pathogen). Globally, the four serotypes of DENV cause an estimated 100 million new cases of dengue fever and 250,000 cases of dengue hemorrhagic fever (DHF) per year. Currently, no specific therapy is available, and DENV vaccines are still in early stages of clinical development. There is a pressing need for the development of reliable surrogate markers of protection for vaccine testing and new approaches for safe, effective vaccines. Recently, using a combination of molecular virology and biophysical methodologies we defined a dominant neutralizing epitope on domain III of the envelope (E) protein of West Nile virus (WNV), a closely related flavivirus. Neutralizing antibodies that recognize this epitope bind with high affinity to conserved WNV-specific amino acids, and block infection at a post-attachment step. For this proposal, we will apply this understanding of the structural basis of antibody neutralization of WNV to the related DENV. Using multiple molecular and biophysical approaches, our collaborative team will study the mechanisms of antibody-mediated neutralization against all four DENV serotypes and develop strategies for novel high-throughput diagnostic assays and E protein- based immunogens. In Specific Aim 1, we will generate panels of mAbs against the E proteins of all four DENV serotypes and test their neutralizing and enhancing potential using a high-throughput flow cytometry- based assay. The most potent neutralizing mAbs will be evaluated for protection in a mouse model of DENV infection. In Specific Aim 2, we will develop mechanistic correlates of antibody protection by defining the cellular stages of antibody blockade using in vitro infection assays and identifying DENV recognition determinants using yeast surface display epitope-mapping. In Specific Aim 3, we will assess the structural correlates of antibody neutralization and enhancement using x-ray crystallography and cryo-electron microscopy. In Specific Aim 4, we will develop epitope-based diagnostic assays and immunogens for DENV. Collectively, these studies may be of significant importance in ongoing and future DENV vaccine development programs.